This competing renewal application is focused on functional proteomics studies of activated protein C (APC), an enzyme that exerts two major, distinct activities - (1) anticoagulant activity by targeting coagulation factors Va and Vllla in reactions requiring APC cofactors and (2) direct anti-apoptotic and anti-inflammatory effects on cells by targeting two APC receptors, protease activated receptor 1 (PAR1) and Endothelial Protein C Receptor (EPCR). Our published work and unpublished preliminary data lead directly to the proposed studies and provide strong support for our hypotheses. We hypothesize that EPCR-dependent effects of APC on cells derive, at least in part, from APC's cleavage at Arg41 in PAR1 in a reaction involving PAR1-specific exosites on APC that differ from factor Va exosites. We hypothesize that PAR1 on cells interacts directly with EPCR via extracellular protein loops and/or via transmembrane helix-helix interactions. We hypothesize that there are additional cofactors or adaptors that influence APC's direct effects on cells. We hypothesize that APC endocytosis is required for APC's anti-apoptotic activity and that endocytosed APC directly exerts intracellular anti-apoptotic activity in cells. We hypothesize that APC exerts antiapoptotic activity by downregulating caspase 3 generation and that APC might act upstream of mitochondrial release of cyt c. In testing these hypotheses, we propose: 1) to expand the mutational framework for understanding APC's enzymatic targeting of its key substrates, 2) to characterize high density lipoprotein (HDL) and factor V as APC cofactors, 3) to clarify mechanisms responsible for APC's direct effects on endothelial cells, and 4) to evaluate APC's novel intracellular anti-apoptotic activity. In related translational research, we propose to assess relationships between thrombotic disease and the APC receptors, PAR1 and EPCR, and between thrombotic disease and plasma levels of the APC lipid cofactor, glucosylceramide. These studies will improve our ability to understand, diagnose and treat thrombotic diseases.